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Pathogenic mycobacteria, including the causative agents of tuberculosis and leprosy, are responsible for considerable morbidity and mortality worldwide. A hallmark of these pathogens is their tendency to establish chronic infections that produce similar pathologies in a variety of hosts. During infection, mycobacteria reside in macrophages and induce the formation of granulomas, organized immune complexes of differentiated macrophages, lymphocytes, and other cells. This review summarizes our understanding of Mycobacterium-host cell interactions, the bacterial-granuloma interface, and mechanisms of bacterial virulence and persistence. In addition, we highlight current controversies and unanswered questions in these areas.

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

In lymphoid tissues coinfected with Mycobacterium avium complex (MAC) and HIV-1, increased viral replication has been observed. This study investigates the role of MAC in perpetuating both infections through the recruitment of monocytes as potential new hosts for bacteria and HIV-1. Increased numbers of macrophages were present in the lymph nodes of patients with dual infection as compared with lymph nodes from HIV(+) patients with no known opportunistic pathogens. In a coculture system, monocyte-derived macrophages were treated with HIV-1 or M. avium and its constituents to further define the mechanism whereby MAC infection of macrophages initiates monocyte migration. Monocyte-derived macrophages treated with bacteria or bacterial products, but not HIV-1, induced a rapid 2- to 3-fold increase in recruitment of monocytes. Pretreatment of the monocytes with pertussis toxin inhibited the migration of these cells, indicating a G protein-linked pathway is necessary for induction of chemotaxis and thus suggesting the involvement of chemokines. Analysis of chemokine mRNA and protein levels from M. avium-treated cultures revealed MAC-induced increases in the expression of IL-8, macrophage-inflammatory protein (MIP)-1alpha, and MIP-1beta with donor-dependent changes in monocyte chemotactic protein-1. Pyrrolidine dithiocarbamate, an antioxidant, inhibited the activation of NF-kappaB and significantly diminished the MAC-induced chemotaxis, concurrently lowering the levels of monocyte chemotactic protein-1 and MIP-1beta. These data demonstrate that MAC induces macrophage production of multiple chemotactic factors via NF-kappaB to promote monocyte migration to sites of MAC infection. In vivo, opportunistic infection may act as a recruitment mechanism in which newly arrived monocytes serve as naive hosts for both MAC and HIV-1, thus perpetuating both infections.

Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines elicited by host macrophages upon challenge with pathogenic mycobacteria. Infection of human peripheral blood mononuclear cells or the murine macrophage cell line J774A-1 with Mycobacterium avium induced activation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and c-Jun N-terminal kinase. U0126, an MEK-specific inhibitor, abrogated M. avium-induced TNF-alpha secretion. Transfection of cells with dominant-negative MEK1 led to the suppression of TNF-alpha release in M. avium-challenged macrophages. M. avium activated p38 MAPK and use of the p38 MAPK inhibitor, SB203580, revealed that the p38 signaling pathway negatively regulates activation of ERK1/2 and release of TNF-alpha. Taken together, these results provide evidence that M. avium-induced TNF-alpha release from macrophages depends on an interplay between the ERK1/2 and the p38 MAPK signaling pathways., (Copyright 2002 Federation of European Microbiological Societies)

The current medical therapy for Mycobacterium avium complex is controversial. American Thoracic Society recommendations advocate empiric therapy with drug susceptibility testing only for clarithromycin. At National Jewish, where we mainly see cases complicated by treatment failure and drug intolerance, we use in vitro susceptibility testing and therapeutic drug monitoring to optimize efficacy and reduce toxicity.

Previously, we have found that immunosuppressive macrophages (M(phi)s) induced by Mycobacterium intracellulare-infection (MI-M(phi)s) required cell contact with target T cells to express their suppressor activity against concanavalin A (Con A)-induced T cell mitogenesis. In this study, we examined the profiles of cell-to-cell interaction of MI-M(phi)s with target T cells. First, MI-M(phi)s displayed suppressor activity in an H-2 allele-unrestricted manner, indicating that MHC molecules are not required for cell contact. The suppressor activity of MI-M(phi)s was reduced markedly by paraformaldehyde fixation or treatment with cytochalasin B or colchicine, indicating that vital membrane functions are required for their suppressor activity. Secondly, the suppressor activity of MI-M(phi)s was independent of cell-to-cell interaction via CD40 ligand/CD40 and M(phi)-derived indoleamine 2,3-dioxygenase, which causes rapid degradation of tryptophan in T cells. Thirdly, precultivation of splenocytes with MI-M(phi)s, allowing cell-to-cell contact, reduced Con A- or anti-CD3 antibody-induced mitogenesis but not phorbol myristate acetate/calcium ionophore A23187-elicited proliferation of T cells. In addition, co-cultivation of T cells with MI-M(phi)s caused marked changes in profiles of the tyrosine phosphorylation of 33 kDa, 34 kDa and 35-kDa proteins and, moreover, the activation of protein kinase C and its translocation to the cell membrane. It thus appears that suppressor signals of MI-M(phi)s, which are transmitted to the target T cells via cell contact, principally cross-talk with the early signalling events before the activation of PKC and/or intracellular calcium mobilization.

The virulence of 5 strains of M. intracellulare and 6 strains of M. avium to mice were examined. The former bacteria were obtained from the patients with mycobacterial pulmonary disease and the latter were from AIDS patients respectively. C57BL/6 (NRAMP-1 susceptible) and its NRAMP-1 congenic mice (resistant) were used to evaluate the virulence of these bacteria. Three of the 5 strains of M. intracelllulare showed a relatively high virulence. They grew in the liver, spleen and lungs of susceptible mice and even in the lungs of resistant mice. On the other hand, none of 6 strains of M. avium could grow in either susceptible or resistant mice. In conclusion, our mouse model may be a useful tool for evaluating the virulence of bacteria isolated from mycobacteriosis patients.

Atypical mycobacterial infections of the middle ear are extremely rare. To our knowledge, only eight cases have been reported in the literature, five of which involve Mycobacterium avium intracellulare. We present a case of culture-proven, M. avium intracellulare otomastoiditis in an 8-year-old boy with common variable immunodeficiency syndrome. The patient clinically presented with pain and otorrhea. The histopathology was marked by acid-fast bacilli-laden histiocytes. Consideration of this entity in the differential diagnosis of chronic, recalcitrant otorrhea can lead to timely diagnosis, treatment, an decreased morbidity., (Copyright 2001 by W.B. Saunders Company)

Background: An upregulation of the cell-cycle associated proteins p53 and p21/Waf1/Cip1 induced by mycobacteria was previously reported. We aimed to evaluate the expression of such proteins in peripheral blood human monocyte cultures infected with strains of different mycobacterial pathogens., Methods: The study relied on the immunocytochemical determination of p53, p21/Waf1/Cipl, bcl-2 and on the Tunel detection of apoptosis in monocytes populations cultured on four-welled chamber slides (10(6) cells/well) infected with Mycobacterium tuberculosis, M. bovis and M. avium for four consecutive days (mycobacterium/monocyte ratio 10:1). The results were expressed as mean values and SD of the percentages of stainings recorded in five fields per slide., Results: The statistical analysis with Fischer test demostrated that at most sampling times the p53 and p21/Waf1/Cip1 expression and the apoptosis index were significantly higher in M. tuberculosis infected cultures than in controls (p<0.05). The M. bovis related picture diverged from the previous one for a lower p53 expression (p<0.05) at all sampling times. The M. avium infected culture values did not diverge significantly from the controls., Conclusions: The p53 and p21/Wafl/Cipl upregulation is compatible with both host defense strategies and pathogen strategies (safeguard of intracellular sanctuaries). The discrepancies among different cultures suggest a direct relationship between p53 activation and mycobacterial ability to enter host cells.

Mycobacterium avium and Mycobacterium intracellulare are closely related organisms and comprise the Mycobacterium avium complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to M. intracellulare and appears to be unique to M. avium. We compared the abilities of M. avium and M. intracellulare to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that M. avium and M. intracellulare were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of M. avium and M. intracellulare were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that M. avium could invade more efficiently than M. intracellulare. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using M. avium and M. intracellulare chromosomal DNAs. In all, 21 genes that were present in M. avium but absent in M. intracellulare were identified, including some that may be associated with the ability of M. avium to invade the intestinal mucosa.

Isolates of Mycobacterium avium-intracellulare (MAI) form multiple colony types named red-opaque, white-opaque, red-transparent (RT), and white-transparent (WT). The newly discovered WT morphotype is multidrug resistant relative to other variants in vitro. To determine whether the WT morphotype occurs in humans, 32 MAI-positive clinical samples from 2 sites were plated directly onto indicator agar without prior passage in vitro. WT was the predominant morphotype in 26 (81%) of these samples and was absent in only 2 samples. WT variants grew better than isogenic RT variants in mouse and human macrophage models of infection, and RT clones that passed through such systems underwent rapid shifts to the WT morphotype. The RT morphotype was heterogeneous with regard to infectivity. In summary, the white morphotype was common in humans and was favored in disease models. It may play an important role in the establishment and persistence of MAI infection.

Systemic human disease caused by organisms of the Mycobacterium avium-Mycobacterium intracellulare complex (MAC) represent a chronic intracellular infection in human hosts who are usually immunocompromised. To develop improved treatment and prophylaxis, and to obtain a better understanding of pathogenesis, we studied the beige mouse (C57 beige(+)/beige(+)) challenged orally or intravenously with a human isolate that causes lethal disease in patients with AIDS (MAC 101, serovar 1). Encouraging anti-MAC studies in animals, as reviewed here, should provide the basis for considering human trials with a promising agent. The ability of an antimicrobial agent to achieve high intracellular concentrations has correlated with the in vivo activity of several specific compounds.

The clinicopathologic spectrum of infections due to nontuberculous mycobacteria (NTM) includes cavitary disease, opportunistic infection, and nodular disease associated with bronchiectasis. We report a less well-described manifestation of NTM infection: 10 immunocompetent patients without preexisting bronchiectasis had radiographic evidence of diffuse infiltrative lung disease. The most common symptoms were dyspnea, cough, hypoxia, and fever. All 10 patients had used a hot tub. Histologic examination revealed exuberant nonnecrotizing, frequently bronchiolocentric, granulomatous inflammation in all cases. In 1 case, necrotizing granulomas were also noted. The inflammation often was associated with patchy chronic interstitial pneumonia and organization. Cultures revealed NTM in all cases (Mycobacterium avium complex in all but 1 case), but staining for acid-fast bacilli was positive in only 1 case. Four patients received corticosteroids alone for presumed hypersensitivity pneumonia, 4 were treated with antimycobacterial therapy, and 2 received both. All patients demonstrated significant improvement at the time of follow-up. These findings suggest that disease due to NTM may manifest as diffuse infiltrates in immunocompetent adults and that hot tub use may be an important risk factor for this disease pattern.

A number of theories regarding the aetiology of Crohn's disease have been proposed. Diet, infections, other unidentified environmental factors and immune disregulation, all working under the influence of a genetic predisposition, have been viewed with suspicion. Many now believe that Crohn's disease is a syndrome caused by several aetiologies. The two leading theories are the infectious and autoimmune theories. The leading infectious candidate is Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of Johne's disease, an inflammatory bowel disease in a variety of mammals including cattle, sheep, deer, bison, monkeys and chimpanzees. The evidence to support M. paratuberculosis infection as a cause of Crohn's disease is mounting rapidly. Technical advances have allowed the identification and/or isolation of M. paratuberculosis from a significantly higher proportion of Crohn's disease tissues than from controls. These methodologies include: (i) improved culture techniques; (ii) development of M. paratuberculosis-specific polymerase chain reaction assays; (iii) development of a novel in situ hybridization method; (iv) efficacy of macrolide and anti-mycobacterial drug therapies; and (v) discovery of Crohn's disease-specific seroreactivity against two specific M. paratuberculosis recombinant antigens. The causal role for M. paratuberculosis in Crohn's disease and correlation of infection with specific stratification(s) of the disorder need to be investigated. The data implicating Crohn's as an autoimmune disorder may be viewed in a manner that supports the mycobacterial theory. The mycobacterial theory and the autoimmune theory are complementary; the first deals with the aetiology of the disorder, the second deals with its pathogenesis. Combined therapies directed against a mycobacterial aetiology and inflammation may be the optimal treatment of the disease.

Mycobacterium avium undergoes reversible morphotypic switching between the virulent transparent colony type and the less virulent opaque colony type. A new morphotypic switch in M. avium, termed red-white, that becomes visible when opaque colonies of clinical isolates are grown on agar media containing Congo red, was recently described. White opaque (WO) variants were found to be more resistant to multiple antibiotics than were red opaque (RO) variants. The present paper reports that transparent derivatives of RO and WO clones retain the differential Congo red binding properties of their opaque parents, indicating that the opaque-transparent switch operates independently of the red-white switch. White transparent variants were more resistant to clarithromycin and rifampin in vitro, and better able to survive within human macrophages, than their red transparent counterparts. Neither red nor white variants were markedly favoured during growth in vitro; however, red variants were better able to spread on soft agar (sliding motility), a potential selective advantage under some environmental circumstances. White-to-red switching was frequently observed in vitro and was accompanied by decreased antibiotic resistance and increased motility. Red-to-white switching has yet to be observed in vitro, indicating that the red morphotype is very stable. Significantly, some widely studied laboratory reference strains of M. avium, including strain 2151 and the genome sequence strain 104, are stable red clones. These strains are intrinsically antibiotic resistant and virulent in animal models, but they may not express genes encoding the elevated levels of antibiotic resistance and intracellular survival observed in white variants.

Mycobacterium avium is a common pathogen in AIDS patients that is primarily (but not exclusively) acquired through the gastrointestinal tract, leading to the development of bacteraemia and disseminated disease. To cause infection through the gut, binding and invasion of the intestinal epithelial barrier are required. To characterize this process further, we determined the cell surface(s) (basolateral vs. apical membrane) that M. avium interacts with in intestinal mucosal cells in vitro. The level of binding and invasion of both HT-29 and Caco-2 intestinal cell monolayers by M. avium were similar when the assay was performed with control medium in the presence of Ca2+ (when only the apical surface was exposed), with Ca2+-depleted medium or with Ca2+-depleted medium + 1 mM EGTA (exposure of both apical and basolateral membranes), suggesting that the bacterium enters the apical surface of the epithelial lining. These observations were confirmed by assays in a transwell system and by using fluorescent microscopy. Real-time video microscopy showed that M. avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated that M. avium invasion is dependent on the activation of the small GTPases Rho, but not on Rac or Cdc42. Passage of M. avium through HT-29 cells led to a phenotypic change (intracellular growth; IG) that was associated with a significantly greater (between five- and ninefold) ability to bind to and invade new monolayers of epithelial cells or macrophages when compared with the invasion by M. avium grown on agar (extracellular growth; EG). IG phenotype invasion of HT-29 cells also takes place only by the apical surface. M. avium enters intestinal epithelial cells by the apical surface and, once within the cells, changes phenotype, becoming more invasive towards both macrophages and other epithelial cells.

Resistance to clarithromycin in breakthrough Mycobacterium avium complex (MAC) isolates typically occurs 3 to 4 months after the initiation of monotherapy in bacteremic AIDS patients. It has been suggested that continuation of clarithromycin therapy still results in clinical and microbiological improvement. To study this paradox, C57BL/6 beige mice were infected with a clarithromycin-resistant (MIC, > or =128 microg/ml) strain of MAC 101 (CLA-R MAC 101) and treated with 200 mg of clarithromycin per kg of body weight/day alone or in combination with ethambutol (100 mg/kg/day) for 2 weeks. Mice infected with a clarithromycin-susceptible strain of MAC 101 had bacterial loads reduced by 90% in the liver and 91% in the spleen (P<0.05, compared with the control). Clarithromycin treatment of CLA-R MAC 101 resulted in a 65% reduction of bacterial loads in the liver (P = 0.009) and a 71% reduction in the spleen (P = 0.009), compared with the results for the untreated control. CLA-R MAC 101 and MAC 101 (isogenic strains) had comparable growth rates in murine tissue, ruling out a loss of virulence of CLA-R MAC 101. Strains of MAC currently defined as macrolide resistant may still respond to treatment with an agent such as clarithromycin within infected tissues.

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Sequence analysis of the ribosomal internal transcribed spacer of 56 Mycobacterium avium complex isolates from pediatric patients with AIDS or lymphadenitis revealed (similar to the situation in adults) that the closely related Mav-B and Mav-A sequevars caused the vast majority of disease. IS1245 restriction fragment-polymorphism analysis and pulsed-field gel electrophoresis revealed sets of isolates with closely related patterns among strains from patients in the Boston area and among isolates from Los Angeles and Miami patients. The finding of related strains that cause disease in epidemiologically unrelated patients is most consistent with one of two hypotheses: (1) a limited subset of M. avium strains is more virulent and therefore more likely to cause disease in humans, or (2) pathogenic strains are more prevalent in the environment.

A total of 738 strains of Mycobacterium avium complex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901 was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901 was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901 positive.

Mycobacterium avium is a common pathogen in AIDS patients and, in a large percentage of those patients, M. avium infection appears to be acquired via the gastrointestinal tract. M. avium is able to bind to and enter human and murine intestinal epithelial cells in vitro and in vivo. The invasion by and intracellular fate of M. avium in the HT-29 intestinal epithelial cell line was examined in an ultrastructural study. Bacterial contact with polarised cells was observed 10-15 min after monolayer infection and in polarised monolayers this always occurred in areas lacking microvilli. Contact with HT-29 cells did not appear to take place in a preferential area on the bacterial cell. Following invasion, M. avium was encountered within vacuoles containing either single or multiple bacteria; the latter evolved to contain only an individual bacterium. Vacuoles containing more than one bacterium were seen early in the infection and eventually underwent segmentation, with each bacterium occupying a vacuole. No bacteria were observed outside vacuoles up to 5 days after infection.

Infections caused by Mycobacterium avium are common in AIDS patients and patients with chronic lung diseases. The bacterium can be acquired both through the intestinal route and respiratory route. M. avium is capable of invading mucosal epithelial cells and translocating across the mucosa. The bacterium can infect macrophages, interfering with several functions of the host cell. The host defense against M. avium is primarily dependent on CD4+ T lymphocytes and natural killer cells. Activated macrophages can inhibit or kill intracellular bacteria by mechanisms that are currently unknown, but M. avium can invade resting macrophages and suppress key aspects of their function by triggering the release of transforming growth factor beta and interleukin 10. Co-infection with HIV-1 appears to be mutually beneficial, with both organisms growing faster.

Mycobacterium avium complex (MAC) is a typical intracellular parasite similar to M. tuberculosis and is one of the most important pathogens that coinfects AIDS patients. Attention has been focused on M. avium infection causing immunosuppression of hosts. Specific serotype-subspecies such as 1, -4 or -8 serotypes can be isolated frequently in humans infected with HIV. Furthermore, the prognosis after infection differs depending on the serotype. Serotype-4 in general shows unfavourable prognosis, while serotype-16 yields rapid recovery. Therefore, we have been interested in the immunomodifying activity of the surface glycopeptidolipid (GPL) antigen. However, no information has been available to date dealing on the virulent factor of MAC that is directly related with intracellular bactericidal activity. Recently, we have tried to test the effect of various GPLs purified from MAC on phagocytic processes of human peripheral blood monocytes (PBMC). We have used GPL-coated heat-killed staphylococcal cells to be phagocytosed by PBMC, and phagosome-lysosome fusion (P-L fusion) was estimated by the acridine orange staining of fused vesicles and bacteria. Results showed strong promotion of phagocytosis and marked inhibition of P-L fusion by serotype-4 GPL, while neither promotion of phagocytosis nor inhibition of P-L fusion in phagocytic cells were shown by serotype-16 GPL. Serotype-8 GPL showed concomitant stimulation of both phagocytosis and P-L fusion. These effects may be due to some unknown interaction between specific carbohydrate chain and organella membranes and serotype-4 GPL may be one of the possible virulent factors in MAC. Comparison with known possible virulent factors such as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate (TMM), glucose 6-monomycolate (GM) or sulfatide was also reported.

Objective: Mycobacterium avium complex (MAC) is isolated with increasing frequency from respiratory specimens. This study was an attempt to determine the significance of this in human immunodeficiency virus (HIV)-positive and HIV-negative patients., Methods: A retrospective cohort study was conducted at Bellevue Hospital, a large municipal hospital in New York City, including all patients with two or more respiratory tract specimens positive for MAC during the period January 1996 to October 1996., Results: Eighty patients met inclusion criteria. Forty-six were HIV-positive, and 34 were HIV-negative. Age, gender distribution, and race were comparable. Cough was a common complaint in all patients, whereas HIV-positive patients were significantly more likely to have fever (19 vs. 2, P < 0.0001). Abnormal chest radiographs were common in both groups (P > 0.8), although HIV-positive patients were more likely to have diffuse abnormalities (P < 0.0001). Focal radiographic findings were similar for both groups; however, there was a trend toward more lymphadenopathy in the HIV-positive group, though this did not reach statistical significance (P = 0.17). Notably, patients in both groups frequently had an established concurrent pulmonary diagnosis or evidence of disseminated MAC infection. Patients who were HIV-positive had Pneumocystis carinii pneumonia (n = 10), pneumonia (n = 10), and disseminated MAC disease (n = 12); whereas the concurrent disease in HIV-negative patients predominantly was active tuberculosis (n = 13). According to the recent American Thoracic Society-recommended criteria for the diagnosis of pulmonary disease caused by nontuberculous mycobacteria only 7 of 46 HIV-positive patients and 1 of 34 HIV-negative patients met clinical, bacteriologic, and radiographic criteria for pulmonary disease caused by MAC (P = 0.052)., Conclusions: Mycobacterium avium complex often is cultured from patients with other lung diseases, and its presence in sputum infrequently signifies true disease, though it is more likely to do so in HIV-positive patients.

Avian tuberculosis accompanied with many tubercular lesions in the liver and spleen was found in a painted quail at a zoological garden in Japan. Mycobacterium avium complex (MAC) serovar 9 without insertion sequence of IS901 was isolated from the liver (1.3 x 10(8) CFU/g), oviduct (9.4 x 10(7) CFU/g), and intestine (1.5 x 10(5) CFU/g). The isolates were inoculated intravenously to chickens. The inoculated chickens showed clinical symptoms of avian tuberculosis. Birds are susceptible to MAC serovar 9 without IS901.

BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Mo) and natural killer (NK) cell numbers increased > or = 4.2 times and T- and B-cell numbers increased > or = 1.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a > or = 31.7-fold increase in Mo and NK cells and a > or = 9.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a > or = 56.7-fold increase in Mo and NK cells and a > or = 22.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Mo and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease.

Mycobacterium avium-intracellulare complex (MAC) is one of the most common opportunistic pathogens in HIV-infected patients. Their synergistic interaction leads to a rapid deterioration of the host defense. In vivo, MAC manifests as a disseminated granulomatous disease that produces a massive inflammatory tissue response perhaps through its activation of inflammatory cytokines. The intracellular signaling following interaction of the mycobacterium with host cells is incompletely understood. Because the response is dependent, in part, on the activation of NF-kappaB, we investigated the effect of MAC on this nuclear transcription factor in cells of macrophage and nonmacrophage lineage. We demonstrate that both high and low virulence strains of MAC potently and rapidly activated NF-kappaB. In supershift assays, using specific Abs against the NF-kappaB subunits, we identified a p50/p65 heterodimer that was formed within 5 min after incubation with the bacterium too rapidly for cytokines to be involved in the activation. This activation was instead mediated through the generation of reactive oxygen intermediates, inasmuch as preincubation of cells with a variety of antioxidants inhibited NF-kappaB activation. Likewise, the transfection of cells with Mn-superoxide dismutase blocked the NF-kappaB activation induced by the bacterium. These data suggest that NF-kappaB activation is a consequence of interaction of host cells with the bacterium and that the interaction may play a pivotal role in the pathogenesis of the disease.

Factors that influence colony morphology are of crucial importance for drug development as well as for understanding the virulence of Mycobacterium avium complex (MAC) strains. The MAC 101 strain used in the present study grows as smooth transparent (SmT) colonies that tend to become opaque and pigmented when incubated for long periods of time. However, when MAC was passaged in animals, two types of colonies were recovered. The new rough transparent (RgT) colony morphology appeared more flat and transparent, having a central spot, irregular edges at times, and a dry, granular appearance like that of the rough mutants. In animal studies, the RgT bacilli multiplied at a much faster rate than that of the SmT bacilli, causing 60-80% mortality compared with the 10% mortality observed in mice infected with SmT. In vitro studies indicated that the SmT MAC did not grow and multiply as well in resident peritoneal macrophages as the RgT MAC did. The two morphotypes did not differ in their growth ratesin vitro but the RgT MAC failed to reduce dimethylthiazol-diphenyltetrazolium bromide (MTT), alamar blue and neutral red, suggesting that there might be significant changes in the cell wall or elsewhere causing changes in cellular permeability. These two morphotypes could serve as models for studying the biochemical markers or the identification of factors responsible for the virulence of the MAC., (Copyright 1998 Academic Press.)

Mycobacterium avium complex (MAC) is one of the most important opportunistic pathogens co-infected with HIV (AIDS) and a typical intracellular parasitic bacteria similar to M. tuberculosis. It is also noticed that M. avium infection causes immunosuppression especially in the cellular immunity of host animals, and specific serotype-subspecies such as sero-2, -4 or -8 can be isolated frequently in human infection. Furthermore, the prognosis after infection differs by the serotypes and serotype-4 shows heavy infection in general, while serotype-16 shows rapid improvement. Therefore, we have been interested in the immunomodifying activity of surface glycopeptidolipid (GPL) antigen. However, to date, no information has been available on the virulence factor of MAC related directly with intracellular bactericidal activity. Recently, we have tested the effect of various GPLs purified form MAC complex on phagocytic processes of human peripheral blood monocytes (PBMC). We have used GPL-coated heat-killed staphylococcal cells to be phagocytosed by PBMC, and phagosome-lysosome (P-L) fusion was estimated by the acridine orange staining of fused vesicles including bacteria. It was revealed that the serotype-4, -12 and -17 GPLs showed strong phagocytosis promotion and marked inhibition of P-L fusion, while serotype-9, -13, -16 and -19 GPLs showed neither promotion of phagocytosis, nor inhibition of P-L fusion in phagocytic cells. Serotype-5, -7, -8 and -10 GPLs showed stimulation of both phagocytosis and P-L fusion, concomitantly. These effects may be due to unknown interaction between specific carbohydrate chain of MAC and phagocytic cell membranes, and serotype-4, -12 and -17 GPLs may be one of the possible virulence factors in MAC.

The capacity of 20 Mycobacterium avium complex isolates to multiply intracellularly in human monocyte-derived macrophages was assessed and correlated to the clinical relevance of each isolate and its reactivity with several candidate genetic virulence markers. The strongest correlation with a virulence phenotype was found for a conserved coding sequence of the macrophage-induced gene mig identified by a specific mig restriction fragment length polymorphism type.

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H2O2 and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H2O2 and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS). Patients with AIDS appear to acquire M. avium mainly through the gastrointestinal tract. Previous studies have shown that healthy mice given M. avium orally develop disseminated infection after 2-4 weeks. The chief site of M. avium invasion of the intestinal mucosa is the terminal ileum. To learn more about the pathophysiology of M. avium infection of the intestinal mucosa, C57BL/6 bg+ bg+ mice were infected orally with M. avium strain 101 and groups of six mice were killed each week for 8 weeks. The terminal ileum was then prepared for histopathological studies and electron microscopy. A delayed inflammatory response was observed and influx of neutrophils in the Peyer's patches was the only abnormality seen at 1 week. A severe inflammatory response was seen from week 2 to week 5 and necrosis of intestinal villi was observed 6 weeks after infection. These results indicate that invasion and infection of the normal intestine by M. avium results in a severe inflammatory response with segmental necrosis of the intestinal mucosa.

Mycobacterium avium complex (MAC) group of microorganisms are the most common opportunistic bacterial pathogens causing disseminated disease in HIV infected patients. These microorganisms are ubiquitous in nature, and are acquired by respiratory and oral routes. Pathogenesis of MAC depends on the ability of the organisms to colonize intestinal/respiratory mucosa, penetrate the protective barriers and resist intracellular killing by macrophages. Transient and reversible variation of colony morphology is one the characteristic feature of MAC that plays a significant role in the virulence and pathogenesis of these microorganisms. Isogenic colony variants of MAC differ in their virulence, susceptibility to antibiotics, stimulation of oxygen radicals and cytokines. The virulent smooth transparent colony variants are more frequently isolated from AIDS patients, more efficient in mucosal colonization, and adhere more efficiently to epithelial cells as compared to the less virulent smooth opaque variants. However, both the isogenic variants bind to the mucosal epithelial cells through the same multiple receptors. In addition, both the isogenic variants of MAC also bind to intestinal mucus through a single receptor. Study of the interaction of MAC with the host cells and characterization of MAC adhesins and host cell receptors facilitates the elucidation of the mechanisms involved in MAC pathogenesis.

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-micron-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.

We tried to characterize the clinical features and findings on chest high resolution computed tomography (HRCT) of patients with Mycobacterium avium-intracellulare (MAI) pulmonary infection without known predisposing lung disease and with no immunodeficiency. We also aimed to clarify the small airway and alveolar inflammation using bronchoalveolar lavage (BAL) from the affected regions. MAI infection was diagnosed in 53 patients from respiratory samples, including sputum and materials obtained using a fiberoptic bronchoscope. None had a predisposing lung disease or immunodeficiency, as assessed by medical history, routine laboratory data, and previously normal chest radiographs and/or CT scans. The mean age of the 53 patients was 60 +/- 11 years, and 48 were nonsmoking females. They had few respiratory symptoms, although 42% had chronic paranasal sinusitis. Chest HRCT findings showed centrilobular small nodules and ectasia of small bronchi and/or bronchioles located mainly in segment (S) 2, 3, 4, and 5. S1, which is usually affected by pulmonary tuberculosis, was completely free of these opacities. The BAL study revealed that the predominant cells were activated T lymphocytes and neutrophils. The CD4+/CD8+ ratio increased significantly. Bacteriology was negative for other bacteria and fungi. Although our patients did not present with distinct respiratory symptoms, the regions affected by MAI showed a chronic inflammation of mainly neutrophils and activated T lymphocytes. The presence of chronic sinusitis may be merely coincidental. However, its high prevalence and the finding of bronchiectasis in chest HRCT raise the question of whether silent bronchiectasis may be a predisposition.

In this study we showed that Mycobacterium avium complex (MAC) clinical isolates differed by the expression of hemolytic activity. Two hemolytic MAC strains were less susceptible to the mycobactericidal effect of murine macrophages than two unhemolytic MAC isolates. In vivo, hemolytic MAC bacilli survived in the spleens of infected mice for a longer time than unhemolytic MAC strains. This suggested a role of hemolysins in the virulence of MAC strains. There was no difference in the cytotoxicity of T cells from mice immunized with M. bovis BCG towards macrophages infected in vitro with MAC strains expressing or not expressing hemolytic activity.

The Mycobacterium avium complex (MAC) includes the closely related species M. avium, M. intracellulare and M. paratuberculosis. The insertion elements IS900, IS901, IS1245 and IS1311 were used as DNA probes to characterize by restriction fragment polymorphisms (RFLPs) eight reference strains, three animal isolates of M. paratuberculosis from outside New Zealand and 61 selected New Zealand MAC isolates from cattle, deer, pigs, sheep and humans. IS900 was found only in strains of M. paratuberculosis. All MAC strains contained IS1311 and the RFLPs associated with this insertion element divided M. paratuberculosis strains into the same groups as IS900 RFLPs. Except for M. paratuberculosis, all MAC strains contained IS1245 and the majority of those from lesions in cattle, deer and pigs also contained IS901. All animal strains containing IS901 had the same RFLPs with IS901, IS1245 and IS1311. In three cases, these apparently identical strains could be differentiated by restriction fragment analysis with BstEII. IS901 was not present in four human isolates or in isolates from deer without lesions. These results indicate that a very closely related group of strains causes the majority of non-paratuberculosis MAC lesions in animals in New Zealand.

Tuberculosis is the leading cause of death due to an infectious organism, killing an estimated 3 million people annually. Mycobacterium tuberculosis, the causative agent of tuberculosis, and other pathogenic mycobacteria require entry into host macrophages to initiate infection. An invasion mechanism was defined that was shared among pathogenic mycobacteria including M. tuberculosis, M. leprae, and M. avium but not by nonpathogenic mycobacteria or nonmycobacterial intramacrophage pathogens. This pathway required the association of the complement cleavage product C2a with mycobacteria resulting in the formation of a C3 convertase. The mycobacteria-associated C2a cleaved C3, resulting in C3b opsonization of the mycobacteria and recognition by macrophages.

One of the most important opportunistic pathogens associated with AIDS is the Mycobacterium avium complex. M. avium infections are found in up to 70% of individuals in advanced stages of AIDS. The deficiency in our knowledge of these mycobacteria presents an obstacle to the development of a rational approach for controlling these life-threatening infections in immunocompromised persons. It is apparent that M. avium can replicate in host macrophages and persist for long periods. During this time, various components, particularly lipids, accumulate in host macrophages and contribute to the ability of this organism to upset the cytokine homeostasis necessary for controlling infections of this type. M. avium lipids are immunosuppressive and can induce a variety of cytokines and eicosanoids that affect general host responses. The intention of this review is to examine the postphagocytic processing of various M. avium lipids with respect to their ability to alter host responses, particularly in immunocompromised patients such as those infected with HIV.

The incidence of identification of nontuberculous mycobacteria has increased since the advent of the acquired immunodeficiency syndrome epidemic. Although Mycobacterium avium complex appears to be responsible for most episodes of nontuberculous disease, several other previously rare species are increasingly being detected, including Mycobacterium kansasii, Mycobacterium fortuitum/chelonei complex, and Mycobacterium genavense. This review briefly summarizes the epidemiology and clinical features of these infections, as well as therapeutic and preventive strategies in immunosuppressed patients with nontuberculous mycobacterial infections. Of clinical relevance, nontuberculous mycobacterial infections are difficult to treat and do not respond to traditional antituberculous agents. The search for more effective treatment regimens is ongoing in an attempt to enhance survival and reduce morbidity among immunocompromised patients. Novel antimicrobial combinations that include clarithromycin or azithromycin have been shown to be effective in treating several nontuberculous mycobacterial infections.

Haemolytic activity of clinical isolates of Mycobacterium bacilli (98) was determined by the method of King et al., 1993. During 3-h incubation, all M. tuberculosis (MTB) isolates (28) and one out of 38 M. avium-intracellulare (MAI) strains, produced a strong contact-dependent haemolysin (CDH). Six MAI strains expressed a weak CDH. One MAI isolate produced a strong and five other MAI strains a weak contact-independent haemolysin (CIH). Two M. bovis BCG strains and 7 M. vaccae strains did not demonstrate haemolytic activity. The persistence of chosen Mycobacterium strains differing by haemolytic activity, in the spleens of infected C57BL/6 mice was examined. Mycobacteria producing a strong CDH (MTB H37Rv, MTB 101/92, MAI 83/93) or CIH (MAI 475/93) survived in the spleens of nonimmunized or M. bovis BCG-immunized mice for longer time than MAI strains expressing weak haemolytic activity or M. bovis BCG vaccine strain.

Mycobacterium avium complex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein of M. avium (Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) of M. leprae (Ml) and M. tuberculosis (Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in an in vitro attachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-alpha by MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-alpha by these cells.

This paper reviews some recent studies which have been performed by us and other investigators, in order to clarify the reason why most mycobacterial infections such as due to Mycobacterium tuberculosis and M. avium complex infections are intractable, that is, why these organisms can escape from attack by microbicidal mechanisms of host macrophages and consequently persist for long time at sites of infection. This paper mainly dealt with the two major subjects, which were studied by using an experimental model for murine M. avium infection. The first subject is on the modes and mechanisms of mycobacterial killing in host macrophages and the mechanisms of bacterial escape from an onslaught by macrophages. The second is on the characteristics of immunosuppressive macrophages induced in M. avium complex infection and the role of the suppressor macrophages in the establishment of immune unresponsiveness of host mice in the progressed stage of infection.